The regulation of the endothelial barrier in blood vessels is a coordinated signaling process that controls the exchange of oxygen and nutrients with the surrounding tissues. Dysregulation of barrier integrity or function is implicated in a range of pathologies, including metabolic disorders such as diabetes (affecting 8.3% of the US population) and cardiovascular disorders such as atherosclerosis (affecting 25% of the US population). Regulation of the barrier is controlled by small gaseous reactive signaling molecules (e.g. nitric oxide and superoxide) that have proven challenging to detect and quantify with adequate selectivity and sensitivity in cells and more importantly in complex tissues and living animals. Imaging has provided significant insight into regulation of the barrier and related physiologic changes that correlate with disease and disease treatment. Yet imaging of signaling molecules associated with the barrier continues to pose significant challenges because the currently available fluorescent biosensors are not sufficiently specific or sensitive to directly report the concentrations and locations of the analytes. Both dye based fluorescent probes and fluorescent protein sensors suffer from limitations that prevent simultaneous correlative measurements of molecular signaling and vascular physiology. In this proposal, we develop a new class of fluorescent molecular biosensor dyes that combine the advantages of indicator dyes with the specificity of genetic encoding. By using tissue specific expression and genetically encoded subcellular targeting, these new biosensors will allow detection of Ca(II), reactive oxygen species (ROS), and reactive nitrogen species (RNS) in specific cells, at specific subcellular locations. These novel targeted fluorescent biosensors are constructed by linking together a sensitive optical sensor of Ca, ROS, or RNS with a fluorescent signaling moiety (FRET acceptor) that is activated upon binding to a genetically encoded receptor, called a fluorogen activating protein (FAP). FAP-bound sensor is able to report (fluorescence signal) the physiology of the sensing at the site of interest. Any biosensors that are not bound to the FAP target are incapable of producing a fluorescence signal and there is no background or non-specific fluorescence to complicate images or analysis of the data. The targeted biosensor dyes will be optimized to work in both cultured endothelial cells and living zebrafish. Transgenic zebrafish will be generated that express the FAP at subcellular locations in specific cells using tissue specific Cre-recombinase expression. We will use these sensors in zebrafish to assess the correlation between Ca(II), ROS and NO signaling, blood flow and barrier function. This project is a close collaboration of three Principal Investigators with distinct expertise at Carnege Mellon University and University of Pittsburgh. Dr. Bruchez is an expert on the development of multichromophore structures for biological detection, and designed the hybrid indicators for biosensing using the fluorogen activating proteins; Dr. St. Croix is an expert on endothelial cell biology RNS/ROS signaling and regulation of endothelial function and more specifically the imaging of endothelium in vitro and in vivo. Dr. Waggoner is an expert in the design of environmentally sensitive dyes, and original developer of the fluorogen activating protein technology.